Introduction. Microbiological diagnosis of bone and joint infections (BJIs) currently relies on standard cultures which are time consuming and lack sensitivity. Various molecular approaches have been described and allowed improvement of BJI diagnosis. This study evaluated for the first time the performance of a DNA microarray-based assay (Prove-it™ Sepsis assay, PISA) for the rapid (<6 hours) detection and identification of 50 different species involved in BJI directly from clinical samples. Material and methods. We retrospectively selected 130 bone and joint samples (67 synovial fluids and 63 bone biopsies) including 114 positive and 16 negative samples. The microbiological diagnosis had been previously established either by culture(C+, n=53) or by PCR16S and sequencing when culture was negative (C-/PCR+). The positive samples were selected to match the species targeted on the DNA microarray. DNA extraction was performed before proceeding to PISA amplification and hybridization on every selected sample. Results. Among the 16 negative samples, one was detected positive with S. epidermidis by PISA, result that was secondarily confirmed using specific PCR. Among the 114 positive samples, 62.3% were positive using PISA with highly concordant identification compared to culture and PCR16S/sequencing results. Forty-three samples (37.7%) remained negative, illustrating a defect of sensitivity. However, PISA accurately detected methicillin resistance not only among the 16 C+/PISA+ Staphylococcus species (n=5) but also among the 28 C-/PCR16S+ Staphylococcus species (n=12) offering crucial rapid information to adapt the treatment of staphylococcal BJIs. Seven polymicrobial samples were also identified without extensive experiments. Discussion – Conclusion. Even if the sensitivity deserves to be improved by optimizing DNA extraction and investigating on human DNA interference, these preliminary promising results highlight that this new and simple microarray method could be in the future an alternative to conventional PCR16S for the diagnosis of BJI

Aims

Occult (clinical) injuries represent 15% of all scaphoid fractures, posing significant challenges to the clinician. MRI has been suggested as the gold standard for diagnosis, but remains expensive, time-consuming, and is in high demand. Conventional management with immobilization and serial radiography typically results in multiple follow-up attendances to clinic, radiation exposure, and delays return to work. Suboptimal management can result in significant disability and, frequently, litigation.

Background. Impaction bone grafting with milled human allograft is the gold standard for replacing lost bone stock during revision hip surgery. Problems surrounding the use of allograft include cost, availability, disease transmission and stem subsidence (usually due to shear failure of the surrounding allograft). Aims. To investigate various polymers for use as substitute allograft. The ideal graft would be a composite with similar mechanical characteristics as allograft, and with the ability to form de novo bone. Methods. High and low molecular weight (MW) forms of three different polymers (polylactic acid (PLA), poly (lactic-co-glycolic) acid (PLGA) and polycaprolactone (PCL)) were milled, impacted into discs, and then tested in a custom built shear testing rig, and compared to allograft. A second stage of the experiment involved the addition of skeletal stem cells (SSC) to each of the milled polymers, impaction, 8 days incubation, and then tests for cell viability and number, via fluorostaining and biochemical (WST-1, DNA) assays. Results. The shear strengths of both high/ low MW PLA, and high/low MW PLGA were significantly higher than those of milled allograft but high and low MW PCL was poor to impact, and had significantly lower shear strengths. Fluorostaining showed good cell survival on high MW PLA, high MW PCL and both high and low MW PLGA. These findings were confirmed on both DNA and WST-1 assays. Conclusions. High MW PLA as well as high and low MW PLGA performed well both in mechanical testing and cell compatibility studies. These three polymers are good contenders to produce a living composite for use as substitute human allograft in impaction bone grafting

INTRODUCTION. Fresh bipolar shell osteochondral allograft (FBOA) is a controversial treatment option for post-traumatic ankle arthritis. Immunological response to transplanted cartilage may play a role in failure. Aim of the study is to compare two groups of patients who received FBOA in association or not to immunosuppressive therapy. METHODS. 2 groups, of 20 patients each, underwent FBOA. Only one group (group-B) received immunosuppressive therapy. Pre-operative and follow-up evaluation were clinical (AOFAS) and radiographical (X-Rays, CT- scan, MRI). Bioptic samples harvested during II look were examined by histochemical, immunohistochemical (ICRS II score) and by genetic typing analyses. RESULTS. Group-A pre-operative AOFAS score improved from 28.2 ± 10.9, to 69.9 ± 18.2 at 24 months follow-up(p<0.005), while Group B improved from 26.2 ± 6.8 to 71.4 ± 7.3 (p<0.005). Comparison of clinical outcomes between the groups was non-significant. Group B showed better morphology of the grafts (ICRS II score mean of 68%) compared to Group A (mean of 40%) (p<0.05). Genetic typing showed a mixed recipient/donor DNA presence. Kendall ordinal correlation between groups and ICRS score was found. All the samples rated as 100 were in group B, while all the samples rated 0 were in group A (=0.506, p=0.008). CONCLUSIONS. Although clinical results were comparable in the two groups, better histological score in Group B evidentiated hyaline cartilage significatively better preserved. Genetic typing showed the presence of cells of the host into the transplanted cartilage suggesting a possible colonization of transplanted cartilage by host cells never described before

The scarcity of mesenchymal stem cells (MSCs) in iliac crest bone marrow aspirate (ICBMA), and the expense and time in culturing cells, has led to the search for alternative harvest sites. The reamer-irrigation-aspirator (RIA) provides continuous irrigation and suction during reaming of long bones. The aspirated contents pass via a filter, trapping bony fragments, before moving into a ‘waste’ bag from which MSCs have been previously isolated. We examined the liquid and solid phases, performed a novel digestion of the solid phase, and made a comparative assessment in terms of number, phenotype and differentiation capacity with matched ICBMA.

The solid fraction from the filtrate was digested for 60 minutes at 37°C with collagenase. Enumeration was performed via the colony-forming unit fibroblast (CFU-F) assay. Passage (P2) cells were differentiated towards osteogenic, adipogenic and chondrogenic lineages, and their phenotypes assessed using flow cytometry (CD33, CD34, CD45, CD73, CD90, and CD105).

MSCs from the RIA phases were able to differentiate at least as well as those from ICBMA, and all fractions had phenotypes consistent with other established sources. The median number of colonies for the three groups was: ICBMA = 8.5 (2 to 86), RIA-liquid = 19.5 (4 to 90), RIA-solid = 109 (67 to 200) per 200 μl. The mean total yield of cells for the three groups was: ICBMA = 920 (0 to 4275), RIA-liquid = 114 983 (16 500 to 477 750), RIA-solid = 12 785 (7210 to 28 475).

The RIA filtrate contains large numbers of MSCs that could potentially be extracted without enzymatic digestion and used for bone repair without prior cell expansion.